氨基免疫荧光探针在瓣膜交联度检测中的应用

Detection of cross-linking degree in artificial aeart valves with amine-specific fluorescent probe

目的利用羧基荧光素琥珀酰亚胺酯(CFSE)能与氨基基团发生特异性不可逆结合,且在波长为488 nm的激发光下发出绿色荧光的特性,检测人工心脏瓣膜的交联程度。方法将去细胞的牛心包浸泡于0.5%的戊二醛溶液中进行交联处理,根据浸泡时间不同分为空白对照组、10 min组、30 min组和6 h组。各组分别制成冰冻切片,经CFSE溶液处理后,通过荧光显微镜观察每组切片的荧光强度,采用图像分析软件计算每组图像的平均荧光强度。同时,从戊二醛处理过的牛心包中提取蛋白质,用聚丙烯酰胺凝胶电泳法(SDS-PAGE)检测各组牛心包的剩余蛋白含量,与氨基免疫荧光探针法进行比较。结果未交联的氨基数量随交联时间增加逐渐减少,荧光强度空白对照组>10 min组>30 min组>6 h组,各组间差异有统计学意义(P<0.01)。聚丙烯酰胺凝胶电泳法检测结果显示,从空白对照组到6 h组可提取的蛋白越来越少。结论与蛋白电泳法检测生物瓣膜交联度相比,CFSE荧光免疫法更为直观灵敏,且能反映瓣膜组织任何部位、层厚的交联效果,为人工生物瓣膜交联度的检测提供新的理论依据和思路。

Objective To detect the cross-linking degree of bioprosthetic valve with carboxyl fluorescein succinimide ester（ CFSE）, which can combine with amino groups specifically and irreversibly and emits green fluorescent in 488 nm excitation optical wavelength. Methods Decellularized bovine pericardium was soaked in 0.5% glutaraldehyde solu- tion and divided into four groups ：blank control group, 10 minutes group, 30 minutes group and 6 hours group. Each specimen was made from frozen section respectively and immersed in the solution of CFSE, as a fluorescent amine-reac- tive probe. The fluorescence image of every frozen section can be observed by fluorescence microscopy and average flu- orescence intensity of each image was measured with image analysis software. At the same time, the protein which was extracted from each group＇s bovine pericardium was tested by SDS-PAGE to compare with the amino immune fluores- cent probe technology. Results The number of cross-linking amino reduced gradually with increased cross-linking time, as reflected in the light intensity of the groups, control group 〉 10 minutes group 〉30 minutes group 〉6 hours group, with remarkable statistics significance among groups （P 〈 0.01 ）. Conclusion Compared with the SDS-PAGE method, amino immune fluorescent probe technology is more intuitive, sensitive, and it can reflect the crosslinking effect of valves organization at any part and any layer. The amino immunefluorescent probe technology may provide a new theoretical basis and train of thought for degree detection of artificial biological valve crosslinking.